Vol. 88: 55-60,1992
MARINE ECOLOGY PROGRESS SERIES
Mar. Ecol. Prog. Ser.
Published October 20
Strictly aerobic and anaerobic bacteria associated
with sinking particulate matter and zooplankton
fecal pellets
Micheline Bianchil, Danielle Martyl, Jean-Louis ~ e y s s i eScott
~ , W. owl er^
'Microbiologie Marine, CNRS UPR 223, Campus de Luminy Case 907, F-13288 Marseilles Cedex 9. France
2 1 ~ Marine
E ~ Environment Laboratory, BP 800, MC-98012 Monaco Cedex
ABSTRACT: Particulate material collected in sediment traps at 80 m depth in coastal northwestern
Mediterranean waters, and fresh fecal pellets of copepods harvested in the surrounding water, were
examined for the presence and activity of ammonia-oxidizing and methanogenic bacteria. Results provide the first evidence for the coexistence of living and active cells of methanogenic and nitrifying bacteria in either fresh zooplankton fecal pellets or large settling particles containing numerous large size
fecal pellets. In addition, the presence of these 2 bacterial types in both copepods and the fecal pellets
produced by them suggests that the bacteria probably originate from the digestive tract of zooplankton, most likely as ingested species for nitrifiers and enteric species for methanogens.
INTRODUCTION
Particulate matenal in ambient seawater provides
microenvironments in which the different chemical
characteristics allow the occurrence of microbial processes that would otherwise be impossible to achieve
on the particle surface or in the surrounding water
(Tranvik & Sieburth 1989).In addition, in these consortia of trophic-specific bacteria embedded within the
same particle, the diverse bacterial groups may interact functionally, one supplying the other with nutrients
(Hobbie & Fletcher 1988).
The vertical distribution of nitrifying bacteria and
their metabolic activity in the oceanic water column
have been shown to be associated with particles (Karl
et al. 1984). The suspended particle layer often coincides with the base of the photic zone and the nitrite
maximum (Spinrad et al. 1989).This layer (between 50
and 150 m) also exhibits a 30 to 70 % supersaturated
CH, concentration relative to atmospheric equilibrium
(Lamontagne et al. 1973, Rudd & Taylor 1980). Deeper
oceanic waters are generally undersaturated, indicating an in situ biological production of methane
(Scranton & Brewer 1977, Sieburth 1987). Methaneproducing bacteria, which are obligate anaerobes, do
O Inter-Research 1992
not exist in a free state in the oxygenated water column, but could survive if associated with reducing
microenvironments such as suspended particles, fecal
pellets or digestive tracts of marine organisms (Oremland 1979, Burke et al. 1983)
The CO-occurrence of ammonia-oxidation and
methane-production activities in particulate material
would furnish evidence for microniches of opposite
redox potential allowing association between ammonia oxidizers and methanogens. A number of marine
species of ammonia oxidizers (reviewed by Bedard &
Knowles 1989) are able to oxidize simultaneously
methane and ammonia, and at the measured open
ocean concentrations, i.e. nanomolar range, ammonia
oxidizers could use methane as a carbon source (Ward
1990).
In order to test this hypothesis, we measured nitrification and methane production in particulate material
collected from the coastal waters off Monaco (1) with
sediment traps at a depth of 80 m in a water column of
200 m deep, and (2) with fresh fecal pellets of copepods
harvested in the surrounding water. The goal of this
initial experiment was not to measure precise quantitative rates of nitrification or methane production in the
different particle types, but to demonstrate the simul-
56
Mar. Ecol. Prog. Ser. 88: 55-60, 1992
taneous occurrence and activities of these 2 bacterial
groups in particulate materials collected in the water
column.
MATERIALS AND METHODS
Sediment trap samples. Unpoisoned sediment traps
were moored from 22 to 25 January 1991 at a site approximately 3 nautical miles off the coast of Monaco.
T h e traps, a cylindrical design with H/D = 2.5 a n d a
collecting area of 0 078 m q F o w l e r et al. 1991), were
moored at 80 m depth in water column approximately
200 m deep. At the e n d of the 3 d collection period, the
contents in the collection cup were placed in a n ice
chest and transported to the laboratory within 2 h of
trap retrieval.
At the laboratory, 1 aliquot of the bulk sample was
immediately removed for the microbial experiments.
Other a l ~ q u o t swere taken to determine mass flux, C
a n d N content a n d the numerical f1u.x of different types
of zooplankton fecal pellets (for technical details, see
Heussner et al. 1987, Fowler et al. 1991, Peinert et al.
1991)
Zooplankton fecal pellets. During the recovery
cruise, zooplankton were collected by making several
horizontal plankton tows (333 pm mesh nets) at approximately 5 m depth at the trap si.te. The zooplankton, principally mixed copepod species, were sequentially sieved through 2000, 1000 and 500 pm mesh
nettin.9 to remove larger species and detritus, and the
fraction retained on 500 pm mesh placed in large shipboard fecal pellet collectors (La Rosa 1976) for return to
the laboratory. The copepods were allowed to defecate
in the collectors for 4 h , after which time 2 fractions of
fecal pellets ( < l 5 0 p m a n d >43 ,um, < 4 3 pm a n d
> 10 pm) were carefully removed from the nets at the
bottom of th.e collector system. These pellets and the
copepods that produced them were immediately used
in the microbial assays.
Nitrifying activity. Nitrification was determined by
measuring the nitnte concentration i.n flasks containing inhibitors (Perfettini & Bianchi 1990). Briefly, in
flasks of 50 m1 final volume, (1) the natural nitrifying
activity was determined by the increase of nitrlte in
flasks witho.ut addition of substrate (NH,); (2) the
potential nitrifying activity was demonstrated by the
incrt'ase of nitkite in duplicates receiving 50 W M final
concentration of NU,Cl. The nitrite concentration corresponded to the increase d u e to ammonia oxidation
minus the decrease d u e to nitrite oxidation. (3) In duplicates with the same concentration of NH,Cl, 10 mM
NaCIOR (final concentration) was added to lnhibit
nitrite oxidation (Belser & Mays 1980). This nitrite
concentration was only d u e to ammonia oxidation. The
difference between the nitrite value in the flasks with
NaC103 and the flasks receiving only NH.,Cl corresponded to the quantity of nitrite disappearing by
possibly several processes including oxidation of
nitrite to nitrate.
All flasks were incubated at 15 "C in the dark, and
nitrite concentra.tions were measured every 2 d over a
period of 50 d. Nitrification rates (pmol nitrite produced I-' d-') were calculated during the exponential
phase of nitrite increase.
For material collected with the sediment trap, 2.2 m1
of the seawater slurry containing particles were used
as a n inoculum for activity measurements and were
transferred lnto 50 m1 (final volume) of 0.2 pm filtered
seawater from the same site.
Following incubation to collect copepod fecal pellets,
the remaining individuals with voided guts were
added to 15 m1 of seawater (<43 pm). Of this water,
3 m1 were used as a n inoculum for activity measurements. Fecal pellets were collected on a 150 pm filter,
the water drained off and the pellets resuspended in
40 m1 of filtered seawater (< 0.2 pm). Of this suspension
2 m1 were then used as a n inoculum. The same protocol was followed for the fecal pellets collected on
43 pm filters.
Methanogenic activity. All experiments were carried out following the methodology of Hungate (1969)
modified for sample collection and ma.nipulation in the
field a n d laboratory. The production of methane (pmol
1-' d - ' ) was assessed by quantifying the increase of
methane in the headspace of tubes containing particulate samples under anoxic conditions with or without
carbon amendments. Methane in the headspace of the
culture tubes was determined every 2 d during a 40 d
incubation period using a Girdel Gas Chromatograph
(Series 30) equipped with a Chromosorb G AWDMCS
column and a flame ionization detector. Argon was
used as the carrier gas, a n d temperatures of column,
injector, and detector were set at 80, 190, and 220 "C,
respectively (Marty et al. 1990).
Eight tubes were inoculated for each pa.rticulate
sample. A sample of 3 m1 of the solution contaming
particles were transferred into 8 tubes supplied with
oxygen-free gas (H2 100 'X,). The natural production of
methane (production from in situ organic material] was
assessed by quantifying methane production in the
headspace of 4 tubes (2 tubes incubated at 15 ' C a n d
2 at 30 "C) without an! amendment. The potential
methane production was measured in the 4 remaining
tubes which r e c e ~ v e da pool of methanoyenic substrates [formate (2.5 g l-', final concentration) + acetate
(2.0 g 1.') + methanol (2.5g l ') + trimethvlamine (2.5g
1 - l ) ] and were incubated at 15 and 30 "C in the dark.
For each sample of particulate material collected
with sediment traps, 5 m1 of the particle slurry
57
Bianchi et al.: Bacteria in particulate matter
were used as inoculum for 8 anaerobic tubes. The
natural and potential production of methane were
then measured at 15 and 30 "C using the previously
described protocol.
After incubation for fecal pellet production, the
'empty' copepods were added to 15 m1 of seawater
( < 4 3 pm) and 3 m1 of this suspension were transferred
into 8 anaerobic tubes. The pellets collected on the
43 and 150 pm filters were resuspended separately in
40 m1 of seawater ( < 0 . 2 pm), and 3 m1 of this suspension were also transferred into 8 anaerobic tubes.
the low end of the range of values that have been
recorded in winter months at this station (Teyssie et al.
1990, Fowler et al. 1991). Nevertheless, the relatively
high fraction (55 %) of the particle mass represented
by fecal pellets is typical for coastal waters in this
region (ibid.).Approximately 70 % of the pellets were
elliptical and of the type normally produced by copepods suggesting that these zooplankton species were
the major contributors to the fecal pellet flux at that
tlme
Nitrifying activity
RESULTS
Sediment trap particles
The characteristics of the sedimenting particulate
material collected off Monaco during that period a r e
presented in Table 1. Both mass flux (ca 0.5 g m-2 d-')
a n d fecal pellet flux (2.6 X 104 pellets m-2 d-') were at
Table 1 Characteristics of particulate material collected in
sediment trap located a t 80 m, 3 miles off Monaco, 22 to 25
January 1991
Sample parameter
Measured value
Total particle dry weight
Mass flux
Total C content
Organic C content
Total N content
Fecal pellet flux
Total pellet no.
115.5 mg
494 mg m-' d - '
6.61 ? 0.01 %
4 17 f 0.01 "%I
0 50 f 0.01 Y,
2 62 X 104 pellets m-' d.'
6120 pellets
Pellet type
Fraction of total pellets
("/.l
Elliptical I
Elliptical I1
Elliptical 111
Cylindrical
Spherical
Salp-like flakes
Amorphous
65.3
1.31
5.23
13.71
9.81
3.93
0.65
Fecal mass contribution to total mass"
Mean volume
(pm3)
4 28x
4 05 X
9.00 X
2 78 X
9 08x
10'
106
107
io7
10"
Not measured
Not measured
- 55%
dCalculations based on formula:
Fecal pellet dry weight =
(mean pellet volume for each type) X (fecal pellet density)
X (number of each fecal pellet type)
(fecal pellet wet weight)/(fecal pellet dry weight)
where fecal density is 1.22 g pm-3 X 10-'' (Komar e t al.
1981). fecal pellet wet weight/dry weight = 4.4
(Fowler 1977)
The particulate material collected in the trap at 80 m
depth demonstrated the highest natural nitrification
rate (Table 2). The addition of ammonium a s substrate
increased more than 10 times the natural nitrifying
activity. When nitrite oxidation was inhibited in the
presence of C103, the measured ammonia oxidation
rate was similar to the potential rate value, demonstrating the absence of nitrite oxidation. Normalized to
the weight of particulate material, oxidation rates were
0.42 a n d 5.84 pm01 g-' d - ' ammonia oxidized during
natural and potential nitrification, respectively. As w e
were not able to separate the different kinds of particles (especially fecal pellets) in the trap sample, it was
not possible to determine whether or not this activity
was related to a specific particle type. From the potential ammonia oxidation, a n d using a coefficient of
0.7 pm01 cell-' d-' (Ward et al. 1982), w e were able to
estimate the presence of 7.9 X 103 cells g-' ammonia
oxidizers.
During the same experiment, samples were also collected at 40 m depth by means of miniaturized sediment traps. This particulate material exhibited a strong
potential activity (up to 38 pm01 nitrite produced 1-'
particle slurry d - l ) with a nitrite oxidation rate which
was about half the ammonia oxidation rate. These
results demonstrated the simultaneous presence of
both ammonia and nitrite oxidizers in these particles.
Unfortunately, detailed characteristics of the particulate material were not determined in this 'mini1-trap,
however, visually the sample appeared to be similar to
that obtained at 80 m .
In incubations with dead animals, the flasks exhibited a growth of heterotrophic bacteria as demonstrated by the turbidity of the water. Ammonium was
probably produced from the decaying organic material, a n d hence the natural nitrifying activity was not
negligible (0.55 pm01 nitrite produced 1-' copepod suspension d - ' ) . Potential rates of nitrification in the copepods were low a n d no nitrite oxidation was detected.
Fecal pellets, which ranged in size from 43 to
150 pm, showed high rates for all activities tested. In
particular, the nitrite oxidation rate (8.70 pm01 1-' fecal
Mar. Ecol. Prog. Ser. 88: 55-60. 1992
58
Table 2. Nitrifying activity expressed as increase or decrease in nitrite concentration (pmol of NO, produced or consumed)
Sample
(B1
Potential
nitnf~cation
(A!
Natural
nitrification
(C)
Potential
ammonia oxidation
(D)
Potential
nitrite oxidation
Sediment trap particles
pm01 l-l particle slurry d-I
pm01 g - ' dry w e ~ g h d-l
t
Copepods
ymoll-' of copepod suspension d-'
0.55
0.32 (1/2)
Fecal pellets
#urn01l-l fecal pellet suspension d - '
< l 5 0 pm and >43 &m
<43 vm and > l 0 pm
1.05
0.12
1.07
0.10
0.27 (1/2)
0
( A ) No substrate added; (B) 50 pM NH4Cl added; (C) nitrite oxidation inhibited: 50 pM NH4CI + 10 mM NaClO, added;
(D) calculated as. rate (C) rate (B)
-
(1/2): Only 1 duplicate positive
pellet suspension d - ' ) was similar to the ammonia oxidation rate (9.77 pm01 I-' suspension d-l). It should be
noted that during similar experiments performed with
copepod fecal pellets harvested in the Gulf of
Marseilles, ammonia oxidation activity was detectable
within the first 5 d of incubation (unpubl.). Table 2 also
shows that the natural activity (1.05 pm01 1-' fecal
pellet suspension d - ' ) was identical to the potential
activity (1.07 ,urn01 1-' suspension d - l ) . The fecal pellets contained both ammonia and nitrite oxidizers
which performed the 2 steps of nitrification at the same
time and at the same rate. This activity, compared with
the absence of activity in 'empty' animals, underscores
the important role of gut microflora in copepods (and
probably other zooplanktonic animals) in controlling
the biogeochemical cycles of elements such as nitrogen through the permanent inoculation of the water
column by specialized bacterial populations.
Fecal pellets in the smaller size class (10 to 43 Km)
exhibited very low nitrifying activity, ranging between
0.10 and 0.12 pm01 nitrite produced 1-' fecal pellet suspension d-'. Nitrite oxidation was also detected, but
the rate was low (0.02 pm01 1-' suspension d-l). Thls
size class of fecal pellets contained less material than
the 43 to 150 pm class, which could explain the lower
activities measured.
Methanogenic activity
Table 3 compares the rates of methane production at
in situ temperature (15 "C) and at 30 "C in the presence
and absence of exogenous substrates. It is important to
note that these results do not represent in situ rates,
because of the long incubation period (40 d) required
for the production of methane; however, they do indicate potential methanogenic activities.
Table 3. Methanogenic activity expressed as increase in methane concentration (pmol of CH, produced)
Sample
15 "C
No addition
30 "C
Sediment trap particles
pm01 1-' particle slurry d - '
pm01 g-l dry weight d - '
0
0
Copepods
pm01 1-' of copepod suspension d.'
0
0.10
Fecal pellets
pm01 I - ' of fecal pellet suspension d - '
< 150 pm and > 4 3 pm
<43 pm and > 10 ,urn
0
0
0
0.05 (1/2)
(1/2):Only 1 duplicate positive
41.27 (1/2)
12.51
Substrate addition
15 "C
30 "C
0.10 (1/2)
0 03
0
0
6.30
0.41
0.13
0.25
219.82 (1/2]
0.17 (1/2)
Bianchi et al.: Bactelria ~n partlculate matter
Methane production was detected in 9 of 16 sets of
duplicate samples, and for 5 of these 9 positive
results, methanogenic activity was observed in only
one of the duplicate tubes. Compared with unsupplemented samples incubated at in situ temperature, in
which methane production was undetectable, both an
increase in incubation temperature and the addition
of substrates caused the expected stimulation of
methanogenic activity. However, the incubation temperature and the presence of substrates have less
effect on methanogenic rates than the high heterogeneity of the particulate material. Therefore, the
dominant factor governing the methane production
rates, both in sediment trap particulates and in copepods and their fecal pellets, appears to be the abundance and/or nature of the particles introduced into
the culture tubes.
The presence of active methanogenic bacteria in
sediment trap particulates has been checked by additional experiments performed on particles collected at
the same sampling station, with miniaturized traps, at
40 and 80 m depth, in which similar high methane production rates (52.80 and 32.81 pm01 CH, produced 1-'
particle slurry d - l , respectively) have been observed.
In these 'minir-traps,which contain a lower abundance
of particulates, methanogenic activity required substrate amendment. This fact suggests that methanogenesis was limited by insufficient amounts of remineralized nutrients released from the particles.
Low, but measurable rates of methane production
were detected in 'empty' zooplankton samples,
whereas fecal pellets exhibited higher rates (Table 3).
Methanogenic activity associated with both copepods
and egested fecal pellets is consistent with the presence of methanogens in the gut of copepods. The differences in methanogenic rates exhibited by the 2 sizefractions (>43 pm and <43 pm) of fecal pellets may be
due to the lower number of pellets in the snlaller sizefraction. Similar experiments (not shown), using copepods collected off Marseilles and their fecal pellets,
have confirmed both the stimulation of methane production by carbon amendment and the presence of
high rates of production in fecal pellets.
DISCUSSION AND CONCLUSIONS
The results reported here provide the first evidence
for the coexistence of methanogenic and nitrifying
bacteria, which exhibit different and incompatible energetic metabolisms, in both fresh zooplankton fecal
pellets and large settling particles. The CO-occurrence
of these 2 types of bacteria could result in an association between the members of this consortium: on one
hand, aerobic nitrifying bacteria which interact with
59
oxygen could contribute, with heterotrophs, to oxygendepleted microzones wherein methanogens can flourish; and on the other hand, anaerobic methanogenic
bacteria would produce methane that could be used as
energy and carbon sources by nitrifiers.
Conrad & Seiler (1988) have observed that elevated
methane concentrations coincided with the accumulation of decaying material. In addition, Ward (1986)
noted a maximum in nitrification rates at the depth of
enhanced nutrient regeneration where fresh organic
matter produces ammonia. The particles we collected
in a sediment trap at 80 m may be considered as fresh
material since the C/N ratio (8.34) was relatively low.
These particulates contained numerous fecal pellets
which have been reported as major zones of decomposition of biological material in the sea (Gowing & Silver
1983).Of the fecal pellets collected at 80 m depth, 79 %
exhibited a mean volume between 2.78 and 4.28 X 107
pm3. The large size of these pellets may allow different, spatially distinct sites to occur in close physical
proximity (Alldredge & Cohen 1987), i.e. some microniches leading to the development of strict aerobes like
nitrifying bacteria, and other microniches favoring the
development of strict anaerobic bacteria such as
methanogens. It is interesting to note that sediment
trap particulates exhibited the highest rates of
methane production (12.51 pm01 g-' d-l) as well as
potential nitrification (5.84 pm01 g - ' d - l ) .
With regard to the presence of nitrifying and
methanogenic bacteria in both copepods and the fecal
pellets produced by them (present results and unpubl.
data), these bacteria probably originate from the
digestive tract of zooplankton and are incorporated
into the fecal pellet interior at the time the pellet is
being produced. The lower bacterial activities
recorded in copepods, compared with those in fecal
pellets, probably resulted from the gut microflora of
copepods being greatly reduced after the passage of
pellets (Sieburth 1991). Bacterial populations inside
fecal pellets could originate as either enteric or
ingested species (Gowing & Silver 1983). Ingested
species may be aerobic bacteria such as the nitrifiers,
which originate from the surrounding seawater and
survive gut passage as digestion resistant forms. In
contrast, enteric bacteria may be anaerobic species,
such as methanogens whose metabolism and growth
are inhibited by even slight traces of oxygen. It should
be further considered that a symbiotic relationship between zooplankton and methanogens is one possible
explanation for the paradoxical production of methane
in the upper ocean (Oremland 1979, Traganza et al.
1979). For example, the anaerobic gut of a zooplankter
provides oxygen-depleted conditions which allow
methanogenic bacteria to develop and, after being
egested in the oxygenated water column, the oxygen-
60
Mar. Ecol. Prog. Ser. 88: 55-60, 1992
depleted microenvironment of fecal material is
maintained by oxygen-consuming bacteria, such as
heterotrophs and nitrifiers.
Acknowledgements The IAEA Marine Environment
Laboratory operates under a bilateral agreement between the
International Atomic Energy Agency and the Government of
the Principality of Monaco. We thank J.-C. Miguel for the C/N
analyses and the crew of the 'Physalie'
Oceanographique) for their assistance with the sample collection.
mu see
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M ~ c r o bEcol. 19: 21 1-225
Manuscript first received: March 20, 1992
Revised version accepted: September 28, 1992